Characterization of analytes found in biological samples is an integral part of both biological research and medical practice. Preparation of samples is a common first step in this process, and varies considerably in complexity depending on the nature of the analyte and the biological sample itself. For determination of the concentration of a therapeutic drug in plasma, sample preparation can be as simple as waiting for a clot to form and transferring a portion of the liquid fraction to an analyzer. Other analytes, such as nucleic acids, may normally be sequestered inside of cells or viruses from which they need to be released prior to characterization. In addition, once released it is often desirable to process such molecules further in order to simplify analysis.
Numerous methods are known for releasing analyte molecules from cells, including mechanical approaches (such as sonication, application of shear forces, application of heat and agitation in the presence of particles) and chemical methods (such as the use of heat (to produce chemical effect), surfactants, chaotropes, and enzymes). Some of these have been adapted for use in cartridge or microfluidics-based platforms such as those represented by single use in-office and point-of-care testing devices and, increasingly, as components of complex systems where contamination is a concern. For example, U.S. Pat. No. 5,874,046A discloses cell disruption through the use of ultrasonics and application of shear forces, among other methods. Similarly, GB2416030B discloses integrated devices that utilizes mechanical or enzymatic digestion to lyse cells and release their contents for analysis. U.S. Pat. No. 6,664,104B2 discloses devices that lyse cells by a variety of means including mechanical disruption, the application of ultrasound, and the addition of chaotropes to the sample. WO2011/119711A1 discloses an assay cartridge that utilizes elevated temperatures and pressures as a sample treatment to release analytes from such biological compartments. Unfortunately, mechanical methods for cell disruption can add significantly to the complexity of an integrated device and the supporting instrumentation. Similarly, addition of chemical reagents such as chaotropes or enzymes, to a sample not only require a means for metering the correct amount of reagent but may require removal of such reagents at a later point in the process lest they complicate analysis. This is particularly true for sensitive analytical methods such as PCR or electrochemistry.
Although it is often used for introduction of foreign genetic material into cells, electroporation has been shown to be an effective means for the release of nucleic acids from cells. Release of nucleic acids from algal cells has been disclosed (Bahi et al, J. R. Soc. Interface, 8:601-608 (2011)), by initially concentrating the cells on an electrode surface and applying a high frequency (600 kHz) current. Unfortunately, such conditions can lead to the production of considerable heat, which may be difficult to dissipate from a microfluidics device without the use of complex heat exchangers or other thermal transfer devices. US2010/0112667A1 discloses the use of a device with a complex electrode configuration that places numerous small insulators placed between a pair of electrodes in order to lyse cells; this configuration generates high current field gradients within the small gaps between the insulators and reduces power requirements. These conditions can also cause other undesirable effects such as bubbles, increased reactive or inhibiting agents, or breakdown of the supporting structures.
Lysis of eukaryotic cells by generation of hydrolysis products within a microfluidic device using electrodes has also been described (Di Carlo et al, Lab Chip, 5:171-178 (2005), wherein local generation of hydroxide ions at relatively high concentrations (estimated at approximately 20 mM) were found to be necessary for rapid lysis. These investigators did not report on recovery or characterization of analytes from the lysed cells. Lee et al (Lab Chip, 10:626-633 (2010)) describe a similar device that incorporates an ion exchange polymer diaphragm to generate high hydroxide concentrations in order to effectively lyse bacterial cells. The authors were able to perform PCR on DNA analytes released in this process but noted that this general approach was not suitable for use with RNA and PCR based detection techniques.
Overall, current methods for release of analytes from cells, viruses, and other biological compartments are difficult to implement in a self-contained microfluidic or point-of-care device. The relatively small size of such devices leads to volume restrictions that complicate operations such as fluid handling related to reagent addition and solid phase capture and release of analytes in order to remove such added reagents prior to analysis. Their reduced dimension also limit options for applying physical forces (such as ultrasound and shear forces) using conventional equipment and provide relatively little heat capacity to control temperature. While electrode-based methods such as electrochemical generation of reactive species have been used, their utility with certain analytes, notably RNA, is unclear. None of these approaches provides controlled analyte fragmentation, which is advantageous in many direct analytical methods, in addition to biological compartment lysis.
The above show that there is an unmet need for a method and device that not only provides reliable, rapid, easily controlled biological compartment lysis and fragmentation of the analytes thus released, but also has simple hardware requirements that are readily adaptable to small scale devices. Such a device and method may be incorporated into a microfluidic “lab on a chip” or point-of-care device where controlled cell lysis and analyte fragmentation has utility.